Negative Feedback Regulation of Wnt4 Signaling by EAF1 and EAF2/U19

Previous studies indicated that EAF (ELL-associated factor) family members, EAF1 and EAF2/U19, play a role in cancer and embryogenesis. For example, EAF2/U19 may serve as a tumor suppressor in prostate cancer. At the same time, EAF2/U19 is a downstream factor in the non-canonical Wnt 4 signaling pathway required for eye development in Xenopus laevis, and along with EAF1, contributes to convergence and extension movements in zebrafish embryos through Wnt maintenance. Here, we used zebrafish embryos and mammalian cells to show that both EAF1 and EAF2/U19 were up-regulated by Wnt4 (Wnt4a). Furthermore, we found that EAF1 and EAF2/U19 suppressed Wnt4 expression by directly binding to the Wnt4 promoter as seen in chromatin immunoprecipitation assays. These findings indicate that an auto-regulatory negative feedback loop occurs between Wnt4 and the EAF family, which is conserved between zebrafish and mammalian. The rescue experiments in zebrafish embryos showed that early embryonic development required the maintenance of the appropriate levels of Wnt4a through the feedback loop. Others have demonstrated that the tumor suppressors p63, p73 and WT1 positively regulate Wnt4 expression while p21 has the opposite effect, suggesting that maintenance of appropriate Wnt4 expression may also be critical for adult tissue homeostasis and prevention against tumor initiation. Thus, the auto-regulatory negative feedback loop that controls expression of Wnt4 and EAF proteins may play an important role in both embryonic development and tumor suppression. Our findings provide the first convincing line of evidence that EAF and Wnt4 form an auto-regulatory negative feedback loop in vivo

Transcriptome Analysis Suggests the Roles of Long Intergenic Non-coding RNAs in the Growth Performance of Weaned Piglets

Long intergenic non-coding RNAs (lincRNAs) have been considered to play a key regulatory role in various biological processes. An increasing number of studies have utilized transcriptome analysis to obtain lincRNAs with functions related to cancer, but lincRNAs affecting growth rates in weaned piglets are rarely described. Although lincRNAs have been systematically identified in various mouse tissues and cell lines, studies of lincRNA in pigs remain rare. Therefore, identifying and characterizing novel lincRNAs affecting the growth performance of weaned piglets is of great importance. Here, we reconstructed 101,988 lincRNA transcripts and identified 1,078 lincRNAs in two groups of longissimus dorsi muscle (LDM) and subcutaneous fat (SF) based on published RNA-seq datasets. These lincRNAs exhibit typical characteristics, such as shorter lengths and lower expression relative to protein-encoding genes. Gene ontology analysis revealed that some lincRNAs could be involved in weaned piglet related processes, such as insulin resistance and the AMPK signaling pathway. We also compared the positional relationship between differentially expressed lincRNAs (DELs) and quantitative trait loci (QTL) and found that some of DELs may play an important role in piglet growth and development. Our work details part of the lincRNAs that may affect the growth performance of weaned piglets and promotes future studies of lincRNAs for molecular-assisted development in weaned piglets

Identification and Functional Prediction of Long Intergenic Non-coding RNAs Related to Subcutaneous Adipose Development in Pigs

An increasing number of studies have shown that long intergenic non-coding RNAs (lincRNAs) are a very important class of non-coding RNAs that plays a vital role in many biological processes. Adipose tissue is an important place for storing energy, but few studies on lincRNAs were related to pig subcutaneous fat development. Here, we used published RNA-seq data from subcutaneous adipose tissue of Italian Large White pigs and identified 252 putative lincRNAs, wherein 34 were unannotated. These lincRNAs had relatively shorter length, lower number of exons, and lower expression level compared with protein-coding transcripts. Gene ontology and pathway analysis indicated that the adjacent genes of lincRNAs were involved in lipid metabolism. In addition, differentially expressed lincRNAs (DELs) between low and high backfat thickness pigs were identified. Through the detection of quantitative trait locus (QTL), DELs were mainly located in QTLs related to adipose development. Based on the expression correlation of DEL genes and their differentially expressed potential target genes, we constructed a co-expression network and a potential pathway of DEL’s effect on lipid metabolism. Our study identified and analyzed lincRNAs in subcutaneous adipose tissue, and results suggested that lincRNAs may be involved in the regulation of subcutaneous fat development. Our findings provided new insights into the biological function of porcine lincRNAs

Characterization and Functional Analysis of Polyadenylation Sites in Fast and Slow Muscles

Many increasing documents have proved that alternative polyadenylation (APA) events with different polyadenylation sites (PAS) contribute to posttranscriptional regulation. However, little is known about the detailed molecular features of PASs and its role in porcine fast and slow skeletal muscles through microRNAs (miRNAs) and RNA binding proteins (RBPs). In this study, we combined single-molecule real-time sequencing and Illumina RNA-seq datasets to comprehensively analyze polyadenylation in pigs. We identified a total of 10,334 PASs, of which 8734 were characterized by reference genome annotation. 32.86% of PAS-associated genes were determined to have more than one PAS. Further analysis demonstrated that tissue-specific PASs between fast and slow muscles were enriched in skeletal muscle development pathways. In addition, we obtained 1407 target genes regulated by APA events through potential binding 69 miRNAs and 28 RBPs in variable 3′ UTR regions and some are involved in myofiber transformation. Furthermore, the de novo motif search confirmed that the most common usage of canonical motif AAUAAA and three types of PASs may be related to the strength of motifs. In summary, our results provide a useful annotation of PASs for pig transcriptome and suggest that APA may serve as a role in fast and slow muscle development under the regulation of miRNAs and RBPs

Analysis of the nicotinamide phosphoribosyltransferase family provides insight into vertebrate adaptation to different oxygen levels during the water-to-land transition

One of the most important events in vertebrate evolutionary history is the water-to-land transition, during which some morphological and physiological changes occurred in concert with the loss of specific genes in tetrapods. However, the molecular mechanisms underlying this transition have not been well explored. To explore vertebrate adaptation to different oxygen levels during the water-to-land transition, we performed comprehensive bioinformatics and experimental analysis aiming to investigate the NAMPT family in vertebrates. NAMPT, a rate-limiting enzyme in the salvage pathway of NAD+ biosynthesis, is critical for cell survival in a hypoxic environment, and a high level of NAMPT significantly augments oxidative stress in normoxic environments. Phylogenetic analysis showed that NAMPT duplicates arose from a second round whole-genome duplication event. NAMPTA existed in all classes of vertebrates, whereas NAMPTB was only found in fishes and not tetrapods. Asymmetric evolutionary rates and purifying selection were the main evolutionary forces involved. Although functional analysis identified several functionally divergent sites during NAMPT family evolution, invitro experimental data demonstrated that NAMPTA and NAMPTB were functionally conserved for NAMPT enzymatic function in the NAD+ salvage pathway. Insitu hybridization revealed broad NAMPTA and NAMPTB expression patterns, implying regulatory functions over a wide range of developmental processes. The morpholino-mediated knockdown data demonstrated that NAMPTA was more essential than NAMPTB for vertebrate embryo development. We propose that the retention of NAMPTB in water-breathing fishes and its loss in air-breathing tetrapods resulted from vertebrate adaptation to different oxygen levels during the water-to-land transition

Ancient duplications and functional divergence in the interferon regulatory factors of vertebrates provide insights into the evolution of vertebrate immune systems

Interferon regulatory factors (IRFs) were first discovered as transcription factors that regulate the transcription of human interferon (IFN)-beta. Increasing evidence shows that they might be important players involved in Adaptive immune system (AIS) evolution. Although numbers of IRFs have been identified in chordates, the evolutionary history and functional diversity of this gene family during the early evolution of vertebrates have remained obscure. Using IRF HMM profile and HMMER searches, we identified 148 IRFs in 11 vertebrates and 4 protochordates. For them, we reconstructed the phylogenetic relationships, determined the synteny conservation, investigated the profile of natural selection, and analyzed the expression patterns in four &quot;living fossil&quot; vertebrates: lamprey, elephant shark, coelacanth and bichir. The results from phylogeny and synteny analysis imply that vertebrate IRFs evolved from three predecessors, instead of four as suggested in a previous study, as results from an ancient duplication followed by special expansions and lost during the vertebrate evolution. The profile of natural selection and expression reveals functional dynamics during the process. Together, they suggest that the 2nd whole-genome duplication (2WGD) provided raw materials for innovation in the IRF family, and that the birth of type-I IFN might be an important factor inducing the establishment of IRF-mediated immune networks. As a member involved in the AIS evolution, IRF provide insights into the process and mechanism involved in the complexity and novelties of vertebrate immune systems. (C) 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license.</p

MEG3 Promotes Differentiation of Porcine Satellite Cells by Sponging miR-423-5p to Relieve Inhibiting Effect on SRF

Although thousands of long noncoding RNAs (lncRNAs) have been identified in porcine growth and development, the regulation mechanisms of functional lncRNAs have not been well explored. In this study, using 5&prime;- and 3&prime;-rapid amplification of cDNA ends (RACE) assays, we obtained two different variants of lncRNA maternally expressed gene 3 (MEG3), namely, MEG3 v1 and MEG3 v2, that were both highly expressed in porcine skeletal muscle and in the early stage of the differentiation of porcine satellite cells. Moreover, we identified the core transcript MEG3 v2. Functional analyses showed that MEG3 overexpression could effectively arrest myoblasts in the G1 phase, inhibit DNA replication, and promote myoblast differentiation, whereas MEG3 knockdown resulted in the opposite effects. Interestingly, the expression of serum response factor (SRF), a crucial transcription factor for myogenesis process, remarkably increased and decreased in mRNA and protein levels with the respective overexpression and knockdown of MEG3. Dual luciferase reporter assay showed that MEG3 could attenuate the decrease of luciferase activity of SRF induced by miR-423-5p in a dose-dependent manner. MEG3 overexpression could relieve the inhibitory effect on SRF and myoblast differentiation induced by miR-423-5p. In addition, results of RNA immunoprecipitation analysis suggested that MEG3 could act as a ceRNA for miR-423-5p. Our findings initially established a novel connection among MEG3, miR-423-5p, and SRF in porcine satellite cell differentiation. This novel role of MEG3 may shed new light on understanding of molecular regulation of lncRNA in porcine myogenesis

Improved genome assembly of Chinese sucker (Myxocyprinus asiaticus) provides insights into the identification and characterization of pharyngeal teeth related maker genes in Cyprinoidei

The Chinese sucker, Myxocyprinus asiaticus (M. asiaticus, Catostomidae, Cypriniformes), is the only living species of Catostomidae in Asia. There are more than 75 species of this family in North America. The fossil record of this group dates back to the early Eocene. As the Chinese sucker is located at the base of the Cyprinoidei phylogeny, this species is also important in clarifying the evolutionary relationships within Cyprinoidei. Here, we assembled a high-quality genome of the Chinese sucker, contig N50 (40.26 ​Mb), which is nearly ten times longer than the previous version (4.19 ​Mb). Phylogenetic analysis identified that Chinese sucker together with Cyprinidae groups are paraphyletic with respect to Cobitoidea. The specific whole genome duplication event of the Chinese sucker was estimated to have occurred ∼25.9 million years ago. Analysis of population historical changes indicated a trend of reduction for the Chinese sucker and T. tibetana. Since Dlx genes play a key role in Cypriniformes pharyngeal teeth development, we conducted a genome-wide identification of Dlx genes, and found that these genes were doubled in whole genome duplication events, followed by the loss of specific copies. Transcriptome results showed that the expression levels of these paralogous genes were similar. This genomic resource provides useful information for the protection of Chinese sucker and functional study of Dlx genes

Identification and Functional Analysis of Long Intergenic Non-coding RNAs Underlying Intramuscular Fat Content in Pigs

Intramuscular fat (IMF) content is an important trait that can affect pork quality. Previous studies have identified many genes that can regulate IMF. Long intergenic non-coding RNAs (lincRNAs) are emerging as key regulators in various biological processes. However, lincRNAs related to IMF in pig are largely unknown, and the mechanisms by which they regulate IMF are yet to be elucidated. Here we reconstructed 105,687 transcripts and identified 1,032 lincRNAs in pig longissimus dorsi muscle (LDM) of four stages with different IMF contents based on published RNA-seq. These lincRNAs show typical characteristics such as shorter length and lower expression compared with protein-coding genes. Combined with methylation data, we found that both the promoter and genebody methylation of lincRNAs can negatively regulate lincRNA expression. We found that lincRNAs exhibit high correlation with their protein-coding neighbors in expression. Co-expression network analysis resulted in eight stage-specific modules, gene ontology and pathway analysis of them suggested that some lincRNAs were involved in IMF-related processes, such as fatty acid metabolism and peroxisome proliferator-activated receptor signaling pathway. Furthermore, we identified hub lincRNAs and found six of them may play important roles in IMF development. This work detailed some lincRNAs which may affect of IMF development in pig, and facilitated future research on these lincRNAs and molecular assisted breeding for pig

Genomic Analysis To Identify Signatures of Artificial Selection and Loci Associated with Important Economic Traits in Duroc Pigs

Identifying genetic basis of domestication and improvement in livestock contributes to our understanding of the role of artificial selection in shaping the genome. Here we used whole-genome sequencing and the genotyping by sequencing approach to detect artificial selection signatures and identify the associated SNPs of two economic traits in Duroc pigs. A total of 38 candidate selection regions were detected by combining the fixation index and the Composite Likelihood Ratio methods. Further genome-wide association study revealed seven associated SNPs that were related with intramuscular fat content and feed conversion ratio traits, respectively. Enrichment analysis suggested that the artificial selection regions harbored genes, such as MSTN, SOD2, MC5R and CD83, which are responsible for economic traits including lean muscle mass, fertility and immunization. Overall, this study found a series of candidate genes putatively associated with the breeding improvement of Duroc pigs and the polygenic basis of adaptive evolution, which can provide important references and fundamental information for future breeding programs